Engineered anti-IL-23 antibodies

ABSTRACT

Engineered antibodies to human IL-23p19 are provided, as well as uses thereof, e.g. in treatment of inflammatory, autoimmune, and proliferative disorders.

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/713,585, filed Aug. 31, 2005, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to interleukin-23 p19 (IL-23p19)specific antibodies and uses thereof. More specifically, the inventionrelates to humanized antibodies that recognize human IL-23p19 andmodulate its activity, particularly in inflammatory, autoimmune andproliferative disorders.

BACKGROUND OF THE INVENTION

The immune system functions to protect individuals from infectiveagents, e.g., bacteria, multi-cellular organisms, and viruses, as wellas from cancers. This system includes several types of lymphoid andmyeloid cells such as monocytes, macrophages, dendritic cells (DCs),eosinophils, T cells, B cells, and neutrophils. These lymphoid andmyeloid cells often produce signaling proteins known as cytokines. Theimmune response includes inflammation, i.e., the accumulation of immunecells systemically or in a particular location of the body. In responseto an infective agent or foreign substance, immune cells secretecytokines which, in turn, modulate immune cell proliferation,development, differentiation, or migration. Immune response can producepathological consequences, e.g., when it involves excessiveinflammation, as in the autoimmune disorders (see, e.g., Abbas, et al.(eds.) (2000) Cellular and Molecular Immunology, W. B. Saunders Co.,Philadelphia, Pa.; Oppenheim and Feldmann (eds.) (2001) CytokineReference, Academic Press, San Diego, Calif.; von Andrian and Mackay(2000) New Engl. J. Med. 343:1020-1034; Davidson and Diamond (2001) NewEngl. J. Med. 345:340-350).

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 andp40 subunits. Studies have indicated that IL-12 plays a critical role inthe differentiation of naïve T cells into T-helper type 1 CD4⁺lymphocytes that secrete IFNγ. It has also been shown that IL-12 isessential for T cell dependent immune and inflammatory responses invivo. (See, e.g., Cua, et al. (2003) Nature 421:744-748. The IL-12receptor is composed of IL-12beta1 and IL-12beta2 subunits.

Interleukin-23 (IL-23) is a heterodimeric cytokine comprised of twosubunits, p19 which is unique to IL-23, and p40, which is shared withIL-12. The p19 subunit is structurally related to IL-6,granulocyte-colony stimulating factor (G-CSF), and the p35 subunit ofIL-12. IL-23 mediates signaling by binding to a heterodimeric receptor,comprised of IL-23R and IL-12β1, which is shared by the IL-12 receptor.A number of early studies demonstrated that the consequences of agenetic deficiency in p40 (p40 knockout mouse; p40KO mouse) were moresevere than those found in a p35KO mouse. Some of these results wereeventually explained by the discovery of IL-23, and the finding that thep40KO prevents expression of IL-12, but also of IL-23 (see, e.g.,Oppmann, et al. (2000) Immunity 13:715-725; Wiekowski, et al. (2001) J.Immunol. 166:7563-7570; Parham, et al. (2002) J Immunol 168:5699-708;Frucht (2002) Sci STKE 2002, E1-E3; Elkins, et al. (2002) InfectionImmunity 70:1936-1948).

Recent studies, through the use of p40 KO mice, have shown that blockadeof both IL-23 and IL-12 is an effective treatment for variousinflammatory and autoimmune disorders. However, the blockade of IL-12through p40, appears to have various systemic consequences such asincreased susceptibility to opportunistic microbial infections.

The most significant limitation in using antibodies as a therapeuticagent in vivo is the immunogenicity of the antibodies. As mostmonoclonal antibodies are derived from rodents, repeated use in humansresults in the generation of an immune response against the therapeuticantibody. Such an immune response results in a loss of therapeuticefficacy at a minimum and a potential fatal anaphylactic response at amaximum. Initial efforts to reduce the immunogenicity of rodentantibodies involved the production of chimeric antibodies, in whichmouse variable regions were fused with human constant regions. Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43. However, mice injectedwith hybrids of human variable regions and mouse constant regionsdevelop a strong anti-antibody response directed against the humanvariable region, suggesting that the retention of the entire rodent Fvregion in such chimeric antibodies may still result in unwantedimmunogenicity in patients.

It is generally believed that complementarity determining region (CDR)loops of variable domains comprise the binding site of antibodymolecules. Therefore, the grafting of rodent CDR loops onto humanframeworks (i.e., humanization) was attempted to further minimize rodentsequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988)Science 239:1534. However, CDR loop exchanges still do not uniformlyresult in an antibody with the same binding properties as the antibodyof origin. Changes in framework residues (FR), residues involved in CDRloop support, in humanized antibodies also are required to preserveantigen binding affinity. Kabat et al. (1991) J. Immunol. 147:1709.While the use of CDR grafting and framework residue preservation in anumber of humanized antibody constructs has been reported, it isdifficult to predict if a particular sequence will result in theantibody with the desired binding, and sometimes biological, properties.See, e.g., Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029,Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181, and Hodgson(1991) Biotechnology (NY) 9:421-5. Moreover, most prior studies useddifferent human sequences for animal light and heavy variable sequences,rendering the predictive nature of such studies questionable. Sequencesof known antibodies have been used or, more typically, those ofantibodies having known X-ray structures, antibodies NEW and KOL. See,e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al.,supra. Exact sequence information has been reported for a few humanizedconstructs. The present invention provides engineered IL-23p19antibodies and uses thereof to treat inflammatory, autoimmune, andproliferative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show comparisons of mouse anti-human IL-23p19 antibody cloneheavy chain variable domain sequences. CDRs are indicated, as are CDRconsensus sequences, for clones 7G10 (SEQ ID NO: 6), 6H12 (SEQ ID NO:5), 13F11 (SEQ ID NO: 7), 13B5 (SEQ ID NO: 8), 7E2 (SEQ ID NO: 33), 13G1(SEQ ID NO: 31), 11C10 (SEQ ID NO: 32), 1E10 (SEQ ID NO: 38), 30F11 (SEQID NO: 34), 34E4 (SEQ ID NO: 35), 5B12 (SEQ ID NO: 42), 6H4 (SEQ ID NO:36), 9C9 (SEQ ID NO: 43), 11B10 (SEQ ID NO: 44), 30E1 (SEQ ID NO: 45),33D2 (SEQ ID NO: 37), 20A9 (SEQ ID NO: 39), 22E9 (SEQ ID NO: 40), 29D5(SEQ ID NO: 41), 21A10 (SEQ ID NO: 9), 33B12 (SEQ ID NO: 10), 10H11 (SEQID NO: 48), 19E9 (SEQ ID NO: 47), 10G8 (SEQ ID NO: 46), 3D7 (SEQ ID NO:14), 39G2 (SEQ ID NO: 11), 35F12 (SEQ ID NO: 12), 49A10 (SEQ ID NO: 13),34F9 (SEQ ID NO: 15), and 7D7 (SEQ ID NO: 16).

FIGS. 2A-2C show comparisons of mouse anti-human IL-23p19 antibody clonelight chain variable domain sequences. CDRs are indicated for clones7G10 (SEQ ID NO: 18), 6H12 (SEQ ID NO: 17), 13F11 (SEQ ID NO: 19), 13B5(SEQ ID NO: 20), 7E2 (SEQ ID NO: 51), 13G1 (SEQ ID NO: 49), 11C10 (SEQID NO: 50), 1E10 (SEQ ID NO: 56), 30F11 (SEQ ID NO: 52), 34E4 (SEQ IDNO: 53), 5B12 (SEQ ID NO: 60), 6H4 (SEQ ID NO: 54), 9C9 (SEQ ID NO: 61),11B10 (SEQ ID NO: 62), 33D2 (SEQ ID NO: 55), 20A9 (SEQ ID NO: 57), 22E9(SEQ ID NO: 58), 29D5 (SEQ ID NO: 59), 21A10 (SEQ ID NO: 21), 33B12 (SEQID NO: 22), 10H11 (SEQ ID NO: 65), 19E9 (SEQ ID NO: 64), 10G8 (SEQ IDNO: 63), 3D7 (SEQ ID NO: 28), 39G2 (SEQ ID NO: 23), 35F12 (SEQ ID NO:24), 49A10 (SEQ ID NO: 25), 34F9 (SEQ ID NO: 26), and 7D7 (SEQ ID NO:27). No light chain variable domain sequence is provided for clone 30E1.Consensus sequences are provided for each of three subfamilies of lightchain CDR sequences, referred to herein as light chain subfamilies (a),(b) and (c). These sequence subfamilies are displayed from top to bottomin FIGS. 2A-2C.

SUMMARY OF THE INVENTION

The present invention is based on the observation that blockade of IL-23inhibits inflammation, autoimmunity, and abnormal proliferation.

The present invention encompasses a binding compound, such as anantibody or fragment thereof, including humanized or chimericrecombinant antibodies, that binds human IL-23p19, comprising at leastone antibody light chain variable region, or binding fragment thereof,having at least one, two or three CDRs selected from the groupconsisting of SEQ ID NOs: 81-89. In one embodiment, the binding compoundof the present invention comprises a light chain variable domaincomprising at least one CDRL1 selected from the group consisting of SEQID NOs: 81-83 and at least one CDRL2 selected from the group consistingof SEQ ID NOs: 84-86 and at least one CDRL3 selected from the groupconsisting of SEQ ID NOs: 87-89.

In one embodiment, the binding compound comprises at least one antibodyheavy chain variable region, or binding fragment thereof, having atleast one, two or three CDRs selected from the group consisting of SEQID NOs: 78-80.

In some embodiments, the binding compound comprises a framework region,wherein the amino acid sequence of framework region is all orsubstantially all of a human immunoglobin amino acid sequence.

In another embodiment, the binding compound of the present inventioncomprises at least one, two or three light chain CDRs having thesequence of SEQ ID NOs: 81-89 or optionally a variant thereof. In oneembodiment, the binding compound comprises at least one, two or threeheavy chain CDRs having the sequence of SEQ ID NOs: 78-80 or optionallya variant thereof. In various embodiments the variant comprises up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservatively modified amino acidresidues relative to the sequence of the respective SEQ ID NOs.Conservative amino acid substitutions are provided at Table 1.

In other embodiments, the binding compound comprises at least oneantibody light chain variable region, or binding fragment thereof,having at least one, two or three CDRs selected from the groupconsisting of SEQ ID NOs: 69-77. In one embodiment, the binding compoundof the present invention comprises a light chain variable domaincomprising at least one CDRL1 selected from the group consisting of SEQID NOs: 69-71 and at least one CDRL2 selected from the group consistingof SEQ ID NOs: 72-74 and at least one CDRL3 selected from the groupconsisting of SEQ ID NOs: 75-77. In one embodiment, the binding compoundcomprises at least one antibody heavy chain variable region, or bindingfragment thereof, having at least one, two or three CDRs selected fromthe group consisting of SEQ ID NOs: 66-68.

In other embodiments, the binding compound of the present inventioncomprises at least one, two or three light chain CDRs having thesequence of SEQ ID NOs: 69-77 or a variant thereof. In anotherembodiment, the binding compound of the present invention comprises alight chain variable domain comprising at least one CDRL1 selected fromthe group consisting of SEQ ID NOs: 69-71 or a variant thereof, and atleast one CDRL2 selected from the group consisting of SEQ ID NOs: 72-74or a variant thereof, and at least one CDRL3 selected from the groupconsisting of SEQ ID NOs: 75-77 or a variant thereof. In one embodiment,the binding compound of the present invention comprises at least one,two or three heavy chain CDRs having the sequence of SEQ ID NOs: 66-68or a variant thereof. In various embodiments the variant comprises up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservatively modified amino acidresidues relative to the sequence of the respective SEQ ID NOs.

In yet another embodiment, the binding compound of the present inventioncomprises at least one, two or three light chain CDRs selected from thegroup consisting of residues 43-53, 69-75 and 108-116 of SEQ ID NOs: 2and 4, and at least one, two or three heavy chain CDRs selected from thegroup consisting of residues 45-54, 69-85 and 118-123 of SEQ ID NOs: 1and 3.

In one embodiment, the binding compound comprises an antibody lightchain variable domain having the sequence of the residues 20-129 of SEQID NO: 2 or 4 or a variant thereof. In one embodiment, the bindingcompound comprises an antibody heavy chain variable domain having thesequence of residues 20-134 of SEQ ID NO: 1 or 3 or a variant thereof.In various embodiments the variant comprises up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40 or 50 or more conservatively modified aminoacid residues relative to the sequence of the respective SEQ ID NOs.

In one embodiment, the binding compound comprises an antibody lightchain comprising, or consisting essentially of, the sequence of themature form (residues 20-233) of SEQ ID NO: 2 or 4. In one embodiment,the binding compound comprises an antibody heavy chain comprising, orconsisting essentially of, the sequence of the mature form (residues20-464) of SEQ ID NO: 1 or 3.

In one embodiment, the binding compound of the present invention bindsto human IL-23p19 (SEQ ID NO: 29) at an epitope comprising residues82-95, or residues 133-140, or both. In another embodiment the IL-23p19binding compound binds to an epitope comprising some or all of residuesE82, G86, S87, D88, T91, G92, E93, P94, S95, H106, P133, S134, Q135,P136, W137, R139 and L140, and optionally residues K83, F90 and L110. Invarious embodiments the epitope for an antibody of interest isdetermined by obtaining an X-ray crystal structure of anantibody:antigen complex and determining which residues on IL-23p19 arewithin a specified distance of residues on the antibody of interest,wherein the specified distance is, e.g., 4 Å or 5 Å. In someembodiments, the epitope is defined as a stretch of 8 or more contiguousamino acid residues along the IL-23p19 sequence in which at least 50%,70% or 85% of the residues are within the specified distance of theantibody.

In other embodiments, the present invention provides a binding compoundthat binds to human IL-23 and has a light chain variable domain (V_(L))with at least 95%, 90%. 85%, 80%, 75% or 50% sequence homology with theresidues 20-129 of SEQ ID NO: 2 or 4. In one embodiment, the presentinvention provides a binding compound that binds to human IL-23 and hasa heavy chain variable domain (V_(H)) with at least 95%, 90%. 85%, 80%,75% or 50% sequence homology with residues 20-134 of SEQ ID NO: 1 or 3.

In one embodiment, the binding compound comprises, or consistsessentially of, an antibody having a light chain having the sequence ofthe mature form (i.e. residues 20-233) of SEQ ID NO: 2 or 4. In oneembodiment, the binding compound comprises, or consists essentially of,an antibody having a heavy chain having the sequence of the mature form(i.e. residues 20-464) of SEQ ID NO: 1 or 3.

In one embodiment, the invention relates to antibodies that are able toblock the binding of a binding compound of the present invention tohuman IL-23 in a cross-blocking assay. In another embodiment, theinvention relates to binding compounds that are able to blockIL-23-mediated activity, such activities including but not limited to,binding to its receptor or promoting the proliferation or survival ofT_(H)17 cells.

In some embodiments, the binding compound of the present inventionfurther comprises a heavy chain constant region, wherein the heavy chainconstant region comprises a γ1, γ2, γ3, or γ4 human heavy chain constantregion or a variant thereof. In various embodiments the light chainconstant region comprises a lambda or a kappa human light chain constantregion.

In various embodiments the binding compounds of the present inventionare polyclonal, monoclonal, chimeric, humanized or fully humanantibodies or fragments thereof. The present invention also contemplatesthat the binding fragment is an antibody fragment selected from thegroup consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and adiabody.

The present invention encompasses a method of suppressing an immuneresponse in a human subject comprising administering to a subject inneed thereof an antibody (or a binding fragment thereof) specific forIL-23 in an amount effective to block the biological activity of IL-23.In some embodiments, the antibody specific for IL-23 is the humanized orchimeric antibody. In further embodiments, the immune response is aninflammatory response including arthritis, psoriasis, and inflammatorybowel disease. In other embodiments, the immune response is anautoimmune response, including multiple sclerosis, uveitis, systemiclupus erythematosus and diabetes. In another embodiment, the immuneresponse is to cancer.

The present invention also contemplates administering an additionalimmunosuppressive or anti-inflammatory agent. The binding compounds ofthe present invention can be in a composition comprising the bindingcompound, or binding fragment thereof, in combination with apharmaceutically acceptable carrier or diluent. In a further embodiment,the composition further comprises an immunosuppressive oranti-inflammatory agent.

The present invention encompasses an isolated nucleic acid encoding thepolypeptide sequence of an antibody embodiment of the binding compoundof the present invention. The nucleic acid can be in an expressionvector operably linked to control sequences recognized by a host celltransfected with the vector. Also encompassed is a host cell comprisingthe vector, and a method of producing a polypeptide comprising culturingthe host cell under conditions wherein the nucleic acid sequence isexpressed, thereby producing the polypeptide, and recovering thepolypeptide from the host cell or medium.

In various embodiments, the invention relates to medicaments comprisingthe binding compounds of the present invention.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. Table 7 belowprovides a listing of sequence identifiers used in this application. Allreferences cited herein are incorporated by reference to the same extentas if each individual publication, patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.

I. Definitions

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compositions derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Proliferative activity” encompasses an activity thatpromotes, that is necessary for, or that is specifically associatedwith, e.g., normal cell division, as well as cancer, tumors, dysplasia,cell transformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an IL-23 agonist or IL-23 antagonist to a humanor animal subject, a cell, tissue, physiological compartment, orphysiological fluid. “Treatment of a cell” also encompasses situationswhere the IL-23 agonist or IL-23 antagonist contacts IL-23 receptor(IL-23R/IL-12Rbeta1 heterodimer), e.g., in the fluid phase or colloidalphase, but also situations where the agonist or antagonist does notcontact the cell or the receptor.

As used herein, the term “antibody” refers to any form of antibody orfragment thereof that exhibits the desired biological activity. Thus, itis used in the broadest sense and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired biologicalactivity.

As used herein, the term “IL-23p19 binding fragment” or “bindingfragment thereof” encompasses a fragment or a derivative of an antibodythat still substantially retain its biological activity of inhibitingIL-23p19 activity. Therefore, the term “antibody fragment” or IL-23p19binding fragment refers to a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules, e.g.,sc-Fv; and multispecific antibodies formed from antibody fragments.Typically, a binding fragment or derivative retains at least 10% of itsIL-23p19 inhibitory activity. Preferably, a binding fragment orderivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or100% (or more) of its IL-23p19 inhibitory activity, although any bindingfragment with sufficient affinity to exert the desired biological effectwill be useful. It is also intended that a IL-23p19 binding fragment caninclude conservative amino acid substitutions that do not substantiallyalter its biologic activity.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., (1991)Nature 352: 624-628 and Marks et al., (1991) J. Mol. Biol. 222: 581-597,for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two V_(H) domains withmodifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The prefix “hum”is added to antibody clone designations when necessary to distinguishhumanized antibodies (e.g. hum6H12) from parental rodent antibodies(e.g. mouse 6H12, or “m6H12”). The humanized forms of rodent antibodieswill generally comprise the same CDR sequences of the parental rodentantibodies, although certain amino acid substitutions may be included toincrease affinity or increase stability of the humanized antibody.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” refers to an antibody which comprises mouse immunoglobulinsequences only.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain; Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, (1987)J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or“FR” residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues. Theresidue numbering above relates to the Kabat numbering system and doesnot necessarily correspond in detail to the sequence numbering in theaccompanying Sequence Listing. See Tables 2 and 3, in which sequencenumbering is with reference to the Sequence Listing.

“Binding compound” refers to a molecule, small molecule, macromolecule,polypeptide, antibody or fragment or analogue thereof, or solublereceptor, capable of binding to a target. “Binding compound” also mayrefer to a complex of molecules, e.g., a non-covalent complex, to anionized molecule, and to a covalently or non-covalently modifiedmolecule, e.g., modified by phosphorylation, acylation, cross-linking,cyclization, or limited cleavage, which is capable of binding to atarget. When used with reference to antibodies, the term “bindingcompound” refers to both antibodies and binding fragments thereof.“Binding” refers to an association of the binding composition with atarget where the association results in reduction in the normal Brownianmotion of the binding composition, in cases where the bindingcomposition can be dissolved or suspended in solution. “Bindingcomposition” refers to a molecule, e.g. a binding compound, incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive, capable of binding to a target.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids are known to those of skill in this artand may be made generally without altering the biological activity ofthe resulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson, et al., Molecular Biology of the Gene, The Benjamin/CummingsPub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions arepreferably made in accordance with those set forth in Table 1 asfollows:

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

The terms “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, that do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a nonlimiting example, a bindingcompound that consists essentially of a recited amino acid sequence mayalso include one or more amino acids, including substitutions of one ormore amino acid residues, that do not materially affect the propertiesof the binding compound.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method route and dose of administration andthe severity of side affects (see, e.g., U.S. Pat. No. 5,888,530 issuedto Netti, et al.). An effective amount can be the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure orparameter by at least 5%, usually by at least 10%, more usually at least20%, most usually at least 30%, preferably at least 40%, more preferablyat least 50%, most preferably at least 60%, ideally at least 70%, moreideally at least 80%, and most ideally at least 90%, where 100% isdefined as the diagnostic parameter shown by a normal subject (see,e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resisteradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from, e.g., a change innumber, change in rate of migration, or change in activation, of cellsof the immune system. Cells of the immune system include, e.g., T cells,B cells, monocytes or macrophages, antigen presenting cells (APCs),dendritic cells, microglia, NK cells, NKT cells, neutrophils,eosinophils, mast cells, or any other cell specifically associated withthe immunology, for example, cytokine-producing endothelial orepithelial cells.

An “IL-17-producing cell” means a T cell that is not a classicalTH1-type T cell or classical TH2-type T cell, referred to as T_(H)17cells. T_(H)17 cells are discussed in greater detail at Cua andKastelein (2006) Nat. Immunol. 7:557-559; Tato and O'Shea (2006) Nature441:166-168; Iwakura and Ishigame (2006) J. Clin. Invest. 116:1218-1222,the disclosures of which are hereby incorporated by reference in theirentireties. “IL-17-producing cell” also means a T cell that expresses agene or polypeptide of Table 10B of U.S. Patent Application PublicationNo. 2004/0219150 (e.g., mitogen responsive P-protein; chemokine ligand2; interleukin-17 (IL-17); transcription factor RAR related; and/orsuppressor of cytokine signaling 3), the disclosure of which is herebyincorporated by reference in its entirety, where expression withtreatment by an IL-23 agonist is greater than treatment with an IL-12agonist, where “greater than” is defined as follows. Expression with anIL-23 agonist is ordinarily at least 5-fold greater, typically at least10-fold greater, more typically at least 15-fold greater, most typicallyat least 20-fold greater, preferably at least 25-fold greater, and mostpreferably at least 30-fold greater, than with IL-12 treatment.Expression can be measured, e.g., with treatment of a population ofsubstantially pure IL-17 producing cells.

Moreover, “IL-17-producing cell” includes a progenitor or precursor cellthat is committed, in a pathway of cell development or celldifferentiation, to differentiating into an IL-17-producing cell, asdefined above. A progenitor or precursor cell to the IL-17 producingcell can be found in a draining lymph node (DLN). Additionally,“IL-17-producing cell” encompasses an IL-17-producing cell, as definedabove, that has been, e.g., activated, e.g., by a phorbol ester,ionophore, and/or carcinogen, further differentiated, stored, frozen,desiccated, inactivated, partially degraded, e.g., by apoptosis,proteolysis, or lipid oxidation, or modified, e.g., by recombinanttechnology.

As used herein, the term “isolated nucleic acid molecule” refers to anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences. Any suitable source ofunrearranged immunoglobulin DNA may be used.

“Inhibitors” and “antagonists” or “activators” and “agonists” refer toinhibitory or activating molecules, respectively, e.g., for theactivation of, e.g., a ligand, receptor, cofactor, a gene, cell, tissue,or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell,is a molecule that alters an activity of the gene, receptor, ligand, orcell, where activity can be activated, inhibited, or altered in itsregulatory properties. The modulator may act alone, or it may use acofactor, e.g., a protein, metal ion, or small molecule. Inhibitors arecompounds that decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate, e.g., a gene, protein, ligand, receptor,or cell. Activators are compounds that increase, activate, facilitate,enhance activation, sensitize, or up regulate, e.g., a gene, protein,ligand, receptor, or cell. An inhibitor may also be defined as acomposition that reduces, blocks, or inactivates a constitutiveactivity. An “agonist” is a compound that interacts with a target tocause or promote an increase in the activation of the target. An“antagonist” is a compound that opposes the actions of an agonist. Anantagonist prevents, reduces, inhibits, or neutralizes the activity ofan agonist. An antagonist can also prevent, inhibit, or reduceconstitutive activity of a target, e.g., a target receptor, even wherethere is no identified agonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activating or inhibiting agent and are compared tocontrol samples without the agent. Control samples, i.e., not treatedwith agent, are assigned a relative activity value of 100%. Inhibitionis achieved when the activity value relative to the control is about 90%or less, typically 85% or less, more typically 80% or less, mosttypically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%. Activation is achieved when theactivity value relative to the control is about 110%, generally at least120%, more generally at least 140%, more generally at least 160%, oftenat least 180%, more often at least 2-fold, most often at least 2.5-fold,usually at least 5-fold, more usually at least 10-fold, preferably atleast 20-fold, more preferably at least 40-fold, and most preferablyover 40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al.(2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Ligand” refers, e.g., to a small molecule, peptide, polypeptide, andmembrane associated or membrane-bound molecule, or complex thereof, thatcan act as an agonist or antagonist of a receptor. “Ligand” alsoencompasses an agent that is not an agonist or antagonist, but that canbind to the receptor. Moreover, “ligand” includes a membrane-boundligand that has been changed, e.g., by chemical or recombinant methods,to a soluble version of the membrane-bound ligand. By convention, wherea ligand is membrane-bound on a first cell, the receptor usually occurson a second cell. The second cell may have the same or a differentidentity as the first cell. A ligand or receptor may be entirelyintracellular, that is, it may reside in the cytosol, nucleus, or someother intracellular compartment. The ligand or receptor may change itslocation, e.g., from an intracellular compartment to the outer face ofthe plasma membrane. The complex of a ligand and receptor is termed a“ligand receptor complex.” Where a ligand and receptor are involved in asignaling pathway, the ligand occurs at an upstream position and thereceptor occurs at a downstream position of the signaling pathway.

“Small molecules” are provided for the treatment of physiology anddisorders of the hair follicle. “Small molecule” is defined as amolecule with a molecular weight that is less than 10 kD, typically lessthan 2 kD, and preferably less than 1 kD. Small molecules include, butare not limited to, inorganic molecules, organic molecules, organicmolecules containing an inorganic component, molecules comprising aradioactive atom, synthetic molecules, peptide mimetics, and antibodymimetics. As a therapeutic, a small molecule may be more permeable tocells, less susceptible to degradation, and less apt to elicit an immuneresponse than large molecules. Small molecules, such as peptide mimeticsof antibodies and cytokines, as well as small molecule toxins aredescribed (see, e.g., Casset, et al. (2003) Biochem. Biophys. Res.Commun. 307:198-205; Muyldermans (2001) J. Biotechnol. 74:277-302; L1(2000) Nat. Biotechnol. 18:1251-1256; Apostolopoulos, et al. (2002)Curr. Med. Chem. 9:411-420; Monfardini, et al. (2002) Curr. Pharm. Des.8:2185-2199; Domingues, et al. (1999) Nat. Struct. Biol. 6:652-656; Satoand Sone (2003) Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482 issuedto Stewart, et a).

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated conditions, a specified ligand binds to a particularreceptor and does not bind in a significant amount to other proteinspresent in the sample.

The antibody, or binding composition derived from the antigen-bindingsite of an antibody, of the contemplated method binds to its antigenwith an affinity that is at least two fold greater, preferably at leastten times greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with unrelatedantigens. In a preferred embodiment the antibody will have an affinitythat is greater than about 10⁹ liters/mol, as determined, e.g., byScatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239).

As used herein, the term “immunomodulatory agent” refers to natural orsynthetic agents that suppress or modulate an immune response. Theimmune response can be a humoral or cellular response. Immunomodulatoryagents encompass immunosuppressive or anti-inflammatory agents.

“Immunosuppressive agents,” “immunosuppressive drugs,” or“immunosuppressants” as used herein are therapeutics that are used inimmunosuppressive therapy to inhibit or prevent activity of the immunesystem. Clinically they are used to prevent the rejection oftransplanted organs and tissues (e.g. bone marrow, heart, kidney,liver), and/or in the treatment of autoimmune diseases or diseases thatare most likely of autoimmune origin (e.g. rheumatoid arthritis,myasthenia gravis, systemic lupus erythematosus, ulcerative colitis,multiple sclerosis). Immunosuppressive drugs can be classified into fourgroups: glucocorticoids cytostatics; antibodies (including BiologicalResponse Modifiers or DMARDs); drugs acting on immunophilins; otherdrugs, including known chemotherpeutic agents used in the treatment ofproliferative disorders. For multiple sclerosis, in particular, theantibodies of the present invention can be administered in conjunctionwith a new class of myelin binding protein-like therapeutics, known ascopaxones.

“Anti-inflammatory agents” or “anti-inflammatory drugs”, is used torepresent both steroidal and non-steroidal therapeutics. Steroids, alsoknown as corticosteroids, are drugs that closely resemble cortisol, ahormone produced naturally by adrenal glands. Steroids are used as themain treatment for certain inflammatory conditions, such as: Systemicvasculitis (inflammation of blood vessels); and Myositis (inflammationof muscle). Steroids might also be used selectively to treatinflammatory conditions such as: rheumatoid arthritis (chronicinflammatory arthritis occurring in joints on both sides of the body);systemic lupus erythematosus (a generalized disease caused by abnormalimmune system function); Sjögren's syndrome (chronic disorder thatcauses dry eyes and a dry mouth).

Non-steroidal anti-inflammatory drugs, usually abbreviated to NSAIDs,are drugs with analgesic, antipyretic and anti-inflammatory effects—theyreduce pain, fever and inflammation. The term “non-steroidal” is used todistinguish these drugs from steroids, which (amongst a broad range ofother effects) have a similar eicosanoid-depressing, anti-inflammatoryaction. NSAIDs are generally indicated for the symptomatic relief of thefollowing conditions: rheumatoid arthritis; osteoarthritis; inflammatoryarthropathies (e.g. ankylosing spondylitis, psoriatic arthritis,Reiter's syndrome); acute gout; dysmenorrhoea; metastatic bone pain;headache and migraine; postoperative pain; mild-to-moderate pain due toinflammation and tissue injury; pyrexia; and renal colic. NSAIDs includesalicylates, arlyalknoic acids, 2-arylpropionic acids (profens),N-arylanthranilic acids (fenamic acids), oxicams, coxibs, andsulphonanilides.

II. General

The present invention provides engineered anti-IL-23 antibodies and usesthereof to treat inflammatory, autoimmune, and proliferative disorders.

A number of cytokines have a role in the pathology or repair ofneurological disorders. IL-6, IL-17, interferon-gamma (IFNgamma), andgranulocyte colony-stimulating factor (GM-CSF) have been associated withmultiple sclerosis (Matusevicius, et al. (1999) Multiple Sclerosis5:101-104; Lock, et al. (2002) Nature Med. 8:500-508). IL-1alpha,IL-1beta, and transforming growth factor-beta 1 (TGF-beta1) plays a rolein ALS, Parkinson's disease, and Alzheimer's disease (Hoozemans, et al.(2001) Exp. Gerontol. 36:559-570; Griffin and Mrak (2002) J. LeukocyteBiol. 72:233-238; Ilzecka, et al. (2002) Cytokine 20:239-243).TNF-alpha, IL-1beta, IL-6, IL-8, interferon-gamma (IFNgamma), and IL-17appear to modulate response to brain ischemia (see, e.g., Kostulas, etal. (1999) Stroke 30:2174-2179; L1, et al. (2001) J. Neuroimmunol.116:5-14). Vascular endothelial cell growth factor (VEGF) is associatedwith ALS (Cleveland and Rothstein (2001) Nature 2:806-819).

Inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis,celiac disease, and irritable bowel syndrome, are mediated by cells ofthe immune system and by cytokines. For example, Crohn's disease isassociated with increased IL-12 and IFNγ, while ulcerative colitis isassociated with increased IL-5, IL-13, and transforming growthfactor-beta (TGFbeta). IL-17 expression may also increase in Crohn'sdisease and ulcerative colitis (see, e.g., Podolsky (2002) New Engl. J.Med. 347:417-429; Bouma and Strober (2003) Nat. Rev. Immunol. 3:521-533;Bhan, et al. (1999) Immunol. Rev. 169:195-207; Hanauer (1996) New Engl.J. Med. 334:841-848; Green (2003) The Lancet 362:383-391; McManus (2003)New Engl. J. Med. 348:2573-2574; Horwitz and Fisher (2001) New Engl. J.Med. 344:1846-1850; Andoh, et al. (2002) Int. J. Mol. Med. 10:631-634;Nielsen, et al. (2003) Scand. J Gastroenterol. 38:180-185; Fujino, etal. (2003) Gut 52:65-70).

Inflammatory diseases of the skin, joints, CNS, as well as proliferativedisorders elicit similar immune responses, thus IL-23 blockade shouldprovide inhibition of these immune mediated inflammatory disorders,without comprising the host ability to fight systemic infections.Antagonizing IL-23 should relieve the inflammation associated withinflammatory bowel disease, Crohn's disease, Ulcerative Colitis,rheumatoid arthritis, psoriatic arthritis, psoriasis, and atopicdermatitis. Use of IL-23 inhibitors will also provide inhibition ofproliferative disorders, e.g., cancer and autoimmune disorders e.g.,multiple sclerosis, type I diabetes, and SLE. Descriptions of IL-23 inthese various disorders can be found in the following published PCTapplications: WO 04/081190; WO 04/071517; WO 00/53631; and WO 01/18051,all of which are incorporated herein by reference.

The p19 subunit of IL-23 is a member of the ‘long chain’ family ofhematopoietic cytokines (Oppmann et al. (2000) supra) and comprises fourpacked α-helices termed A, B, C and D, with an up-up-down-down topology.The 4 helices are connected by 3 polypeptide loops. The A-B and C-Dloops are modeled to be relatively long as they connect parallelhelices. The short B-C loop connects the antiparallel B and C helices.The p19 subunit of IL-23 is a member of the 1-6 family of helicalcytokines. This family of cytokines bind to their cognate receptorsthrough three conserved epitopes (site I, II and III; Bravo and Heath(2000) EMBO J. 19:2399-2411). The p19 subunit interacts with threecytokine receptor subunits to form the competent signaling complex. Whenexpressed in a cell, the p19 subunit first form a complex with the p40subunit, which it shares with IL-12. As noted above, the p19p40 complexis secreted from the cell as a heterodimeric protein and is called IL-23(See, e.g., Oppmann et al., supra). The cellular receptor complexrequired to transduce the IL-23 signal consists of two members of thetall signaling receptor subunits of the IL-6/IL-12 family of cytokines,the IL-23-specific IL-23R (see, e.g., Parham et al. supra) and theIL-12Rb1, that is shared with IL-12.

Insights into the structural basis of ‘long chain’ cytokine/receptorrecognition have shown that although large areas of protein surface areburied in formation of cytokine—receptor complexes, the affinity of theinteraction is dominated by a few, often tightly clustered amino acidresidues forming an energetic ‘hot spot’ in the center of the bindinginterface. The identity of the residues that dominate the binding energyof a large protein-protein interface has been termed the ‘functionalepitope’. The affinity of the interaction (and hence biologicalspecificity) is consequently defined by the structural complementarityof the functional epitopes of ligand and receptor. Detailed mutagenesisstudies have shown that the most significant residues that make up thefunctional epitopes of cytokines and receptors are hydrophobic contactsinvolving either non-polar side chains such as tryptophan, the aliphaticcomponents of non-polar side chains or the polypeptide backbone. Thenon-polar ‘core’ is surrounded by a halo of polar residues of lesserimportance for binding energy. Kinetic studies indicate that the primaryrole of the functional epitopes is to stabilize protein-proteininteraction by decreasing the dissociation rate of the complex. It hasbeen suggested that the initial contact between cytokine and receptor isdominated by random diffusion or ‘rolling’ of protein surfaces producingmany unstable contacts. The complex is then stabilized when thefunctional epitopes of the receptor and ligand engage (see, e.g., Bravoand Heath, supra).

III. Generation of IL-23 Specific Antibodies

Any suitable method for generating monoclonal antibodies may be used.For example, a recipient may be immunized with a linked or unlinked(e.g. naturally occurring) form of the IL-23 heterodimer, or a fragmentthereof. Any suitable method of immunization can be used. Such methodscan include adjuvants, other immunostimulants, repeated boosterimmunizations, and the use of one or more immunization routes.

Any suitable source of IL-23 can be used as the immunogen for thegeneration of the non-human antibody, specific for the p 19 subunit, ofthe compositions and methods disclosed herein. Such forms include, butare not limited whole protein, including linked and naturally occurringheterodimers, peptide(s), and epitopes, generated through recombinant,synthetic, chemical or enzymatic degradation means known in the art.

Any form of the antigen can be used to generate the antibody that issufficient to generate a biologically active antibody. Thus, theeliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents known in the art. The eliciting antigen may be anisolated full-length protein, a cell surface protein (e.g., immunizingwith cells transfected with at least a portion of the antigen), or asoluble protein (e.g., immunizing with only the extracellular domainportion of the protein). The antigen may be produced in a geneticallymodified cell. The DNA encoding the antigen may genomic or non-genomic(e.g., cDNA) and encodes at least a portion of the extracellular domain.As used herein, the term “portion” refers to the minimal number of aminoacids or nucleic acids, as appropriate, to constitute an immunogenicepitope of the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

Any suitable method can be used to elicit an antibody with the desiredbiologic properties to inhibit IL-23. It is desirable to preparemonoclonal antibodies (mAbs) from various mammalian hosts, such as mice,rodents, primates, humans, etc. Description of techniques for preparingsuch monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York,N.Y. Thus, monoclonal antibodies may be obtained by a variety oftechniques familiar to researchers skilled in the art. Typically, spleencells from an animal immunized with a desired antigen are immortalized,commonly by fusion with a myeloma cell. See Kohler and Milstein (1976)Eur. J. Immunol. 6:511-519. Alternative methods of immortalizationinclude transformation with Epstein Barr Virus, oncogenes, orretroviruses, or other methods known in the art. See, e.g., Doyle, etal. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE:LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y. Coloniesarising from single immortalized cells are screened for production ofantibodies of the desired specificity and affinity for the antigen, andyield of the monoclonal antibodies produced by such cells may beenhanced by various techniques, including injection into the peritonealcavity of a vertebrate host. Alternatively, one may isolate DNAsequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according, e.g.,to the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al., Science246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989). Thepolypeptides and antibodies of the present invention may be used with orwithout modification, including chimeric or humanized antibodies.Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,magnetic particles, and the like. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced, see Cabilly U.S. Pat. No. 4,816,567;and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; ormade in transgenic mice, see Mendez et al. (1997) Nature Genetics15:146-156; also see Abgenix and Medarex technologies.

Antibodies or binding compositions against predetermined fragments ofIL-23 can be raised by immunization of animals with conjugates of thepolypeptide, fragments, peptides, or epitopes with carrier proteins.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective IL-23. These monoclonal antibodies will usually bind with atleast a K_(d) of about 1 μM, more usually at least about 300 nM,typically at least about 30 nM, preferably at least about 10 nM, morepreferably at least about 3 nM or better, usually determined by ELISA.Suitable non-human antibodies may also be identified using the biologicassays described in Example 5, below.

IV. Humanization of IL-23 Specific Antibodies

Any suitable non-human antibody can be used as a source for thehypervariable region. Sources for non-human antibodies include, but arenot limited to, murine, Lagomorphs (including rabbits), bovine, andprimates. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance of thedesired biological activity. For further details, see Jones et al.(1986) Nature 321:522-525; Reichmann et al. (1988) Nature 332:323-329;and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

Methods for recombinantly engineering antibodies have been described,e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S.Pat. No. 4,816,567), Law et al. (European Patent Application PublicationNo. 438 310) and Winter (European Patent Application Publication No.239400).

Amino acid sequence variants of humanized anti-IL-23 antibody areprepared by introducing appropriate nucleotide changes into thehumanized anti-IL-23 antibody DNA, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions intoand/or substitutions of, residues within the amino acid sequences shownfor the humanized anti-IL-23 F(ab) (e.g. as in SEQ ID NOs: 1 and 2). Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the humanized anti-IL-23 antibody, suchas changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-IL-23p19 antibody polypeptide that are preferredlocations for mutagenesis is called “alanine scanning mutagenesis,” asdescribed by Cunningham and Wells (1989) Science 244: 1081-1085. Here, aresidue or group of target residues are identified (e.g., chargedresidues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutralor negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with IL-23antigen. The amino acid residues demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, Ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed humanized anti-IL-23p19 antibody variants are screened for thedesired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-IL-23 antibody with an N-terminal methionyl residue orthe antibody fused to an epitope tag. Other insertional variants of thehumanized anti-IL-23 antibody molecule include the fusion to the N- orC-terminus of humanized anti-IL-23 antibody of an enzyme or apolypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanizedanti-IL-23p19 antibody molecule removed and a different residue insertedin its place. The sites of greatest interest for substitutionalmutagenesis include the hypervariable loops, but FR alterations are alsocontemplated. Hypervariable region residues or FR residues involved inantigen binding are generally substituted in a relatively conservativemanner.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Yet another type of amino acid variant is the substitution of residuesto provide for greater chemical stability of the final humanizedantibody. For example, an asparagine (N) residue may be changed toreduce the potential for formation of isoaspartate at any NG sequenceswithin a rodent CDR. In one embodiment, the asparagine is changed toglutamine (Q). Isoaspartate formation may debilitate or completelyabrogate binding of an antibody to its target antigen. Presta (2005) J.Allergy Clin. Immunol. 116:731 at 734. In addition, methionine residuesin rodent CDRs may be changed to reduce the possibility that themethionine sulfur would oxidize, which could reduce antigen bindingaffinity and also contribute to molecular heterogeneity in the finalantibody preparation. Id. In one embodiment, the methionine is changedto alanine (A). Antibodies with such substitutions are subsequentlyscreened to ensure that the substitutions do not decrease IL-23p19binding affinity to unacceptable levels.

Nucleic acid molecules encoding amino acid sequence variants ofhumanized IL-23 specific antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofhumanized anti-IL-23p19 antibody.

Ordinarily, amino acid sequence variants of the humanized anti-IL-23antibody will have an amino acid sequence having at least 75% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,and most preferably at least 95%. Identity or homology with respect tothis sequence is defined herein as the percentage of amino acid residuesin the candidate sequence that are identical with the humanizedanti-IL-23 residues, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, theantibody is an IgG antibody. Any isotype of IgG can be used, includingIgG₁, IgG₂, IgG₃, and IgG₄. Variants of the IgG isotypes are alsocontemplated. The humanized antibody may comprise sequences from morethan one class or isotype. Optimization of the necessary constant domainsequences to generate the desired biologic activity is readily achievedby screening the antibodies in the biological assays described below.

Likewise, either class of light chain can be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

Any suitable portion of the CDR sequences from the non-human antibodycan be used. The CDR sequences can be mutagenized by substitution,insertion or deletion of at least one residue such that the CDR sequenceis distinct from the human and non-human antibody sequence employed. Itis contemplated that such mutations would be minimal. Typically, atleast 75% of the humanized antibody residues will correspond to those ofthe non-human CDR residues, more often 90%, and most preferably greaterthan 95%.

Any suitable portion of the FR sequences from the human antibody can beused. The FR sequences can be mutagenized by substitution, insertion ordeletion of at least one residue such that the FR sequence is distinctfrom the human and non-human antibody sequence employed. It iscontemplated that such mutations would be minimal. Typically, at least75% of the humanized antibody residues will correspond to those of thehuman FR residues, more often 90%, and most preferably greater than 95%.

CDR and FR residues are determined according to the standard sequencedefinition of Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda Md.(1987). SEQ ID NOs: 5-16 and 31-48 show the heavy chain variable domainsequences of various mouse anti-human IL-23p19 antibodies, and SEQ IDNOs: 17-28 and 49-65 depict the light chain variable domain sequences.SEQ ID NOs: 66-68 are consensus sequences for heavy chain CDRs (CDRH1,CDRH2 and CDRH3), and are comprised of the most common amino acidresidue at each position in the heavy chain CDRs for the family ofantibodies consisting of 7G10, 6H12, 13F11, 13B5, 7E2, 13G1, 11C10,1E10, 30F11, 34E4, 5B12, 6H4, 9C9, 11B10, 30E1, 33D2, 20A9, 22E9, 29D5,21A10 and 33B12. FIGS. 1A-1C provide a sequence lineup of heavy chainsof various antibodies of the present invention.

As shown in FIGS. 2A-2C, the light chain CDRs of the antibodies of thepresent invention disclosed herein are grouped into three subfamilies,referred to as (a), (b) and (c). Light chain subfamily (a) consists ofantibodies 7G10, 6H12, 13F11, 13B5, 7E2, 13G1, 11C10, 30F11, 34E4, 6H4,33D2 and 33B12. Light chain subfamily (b) consists of antibodies 1E10,20A9, 22E9, 29D5, 5B12, 9C9 and 11B10. Light chain subfamily (c)consists of antibodies 10H11, 19E9, 10G8, 39G2, 35F12, 49A10, 34F9 and7D7. These light chain subfamilies were used to derive consensus CDRsequences of CDRL1(a), CDRL1(b) and CDRL1(c) (SEQ ID NOs: 69-71) andcorresponding consensus sequences CDRL2 (SEQ ID NOs: 72-74) and CDRL3(SEQ ID NOs: 75-77) for each subfamily. Consensus sequences for lightchain CDRs are comprised of the most common amino acid residue at eachposition in the light chain CDRs for each subfamily of antibodies.

Tables 2 and 3 define various domains of humanized anti-IL-23p19antibodies 6H12, 7G10, 10H11 and 22E9, as well as and the light andheavy chain variable domains of several murine antibodies of the presentinvention. Residues 1-19 of SEQ ID NOs: 1-4 represent signal sequencesfor heavy and light strands of hum6H12 and hum7G10. Light chain constantdomains of hum6H12 and hum7G10 are at residues 130-233 of SEQ ID NOs: 2and 4, respectively. Heavy chain constant domains of hum6H12 and hum7G10 are at residues 135-464 of SEQ ID NOs: 1 and 3, respectively, withCH1 at residues 135-242, CH2+hinge at residues 243-357 and CH3 atresidues 358-464. All other antibodies are presented as light and heavychain variable regions (V_(L) and V_(H)), and thus lack signal sequencesand constant domains.

TABLE 2 Light Chain Sequences and Domains ANTIBODY SEQ ID V_(L) LIGHTCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-L1 CDR-L2 CDR-L3 hum6H12 220-129  43-53 69-75 108-116  hum7G10 4 20-129  43-53 69-75 108-116 hum10H11 91 1-114 24-38 54-60 93-101 hum22E9 93 1-116 24-40 56-62 95-103m6H12 LC 17 1-108 24-34 50-56 89-97  m7G10 LC 18 1-108 24-34 50-5689-97  m13F11 LC 19 1-108 24-34 50-56 89-97  m13B5 LC 20 1-108 24-3450-56 89-97  m21A10 LC 21 1-108 24-34 50-56 89-97  m33B12 LC 22 1-10824-34 50-56 89-97  m39G2 LC 23 1-112 24-38 54-60 93-101 m35F12 LC 241-112 24-38 54-60 93-101 m49A10 LC 25 1-112 24-38 54-60 93-101 m34F9 LC26 1-112 24-38 54-60 93-101 m7D7 LC 27 1-112 24-38 54-60 93-101 m3D7 LC28 1-108 24-34 50-56 89-97  m13G1 LC 49 1-108 24-34 50-56 89-97  m11C10LC 50 1-108 24-34 50-56 89-97  m7E2 LC 51 1-108 24-34 50-56 89-97 m30F11 LC 52 1-108 24-34 50-56 89-97  m34E4 LC 52 1-108 24-34 50-5689-97  m6H4 LC 54 1-108 24-34 50-56 89-97  m33D2 LC 55 1-108 24-34 50-5689-97  m1E10 LC 56 1-114 24-40 56-62 95-103 m20A9 LC 57 1-114 24-4056-62 95-103 m22E9 LC 58 1-114 24-40 56-62 95-103 m29D5 LC 59 1-11424-40 56-62 95-103 m5B12 LC 60 1-114 24-40 56-62 95-103 m9C9 LC 61 1-11424-40 56-62 95-103 m11B10 LC 62 1-114 24-40 56-62 95-103 m10G8 LC 631-112 24-38 54-60 93-101 m19E9 LC 64 1-112 24-38 54-60 93-101 m10H11 LC65 1-112 24-38 54-60 93-101

TABLE 3 Heavy Chain Sequences and Domains ANTIBODY SEQ ID V_(H) HEAVYCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-H1 CDR-H2 CDR-H3 hum6H12 120-134  45-54 69-85 118-123  hum7G10 3 20-134  45-54 69-85 118-123 hum10H11 90 1-118 26-35 50-66 99-107 hum22E9 92 1-115 26-35 50-66 99-104m6H12 5 1-115 26-35 50-66 99-104 m7G10 6 1-115 26-35 50-66 99-104 m13F117 1-115 26-35 50-66 99-104 m13B5 8 1-116 26-35 50-66 99-105 m21A10 91-115 26-35 50-66 99-104 m33B12 10 1-115 26-35 50-66 99-104 m39G2 111-118 26-35 50-66 99-107 m35F12 12 1-118 26-35 50-66 99-107 m49A10 131-119 26-35 50-66 99-108 m3D7 14 1-122 26-35 50-66 99-111 m34F9 15 1-12426-35 50-66 99-113 m7D7 16 1-124 26-35 50-66 99-113 m13G1 31 1-115 26-3550-66 99-104 m11C10 32 1-115 26-35 50-66 99-104 m7E2 33 1-115 26-3550-66 99-104 m30F11 34 1-115 26-35 50-66 99-104 m34E4 35 1-115 26-3550-66 99-104 m6H4 36 1-115 26-35 50-66 99-104 m33D2 37 1-115 26-35 50-6699-104 m1E10 38 1-115 26-35 50-66 99-104 m20A9 39 1-115 26-35 50-6699-104 m22E9 40 1-115 26-35 50-66 99-104 m29D5 41 1-115 26-35 50-6699-104 m5B12 42 1-115 26-35 50-66 99-104 m9C9 43 1-115 26-35 50-6699-104 m11B10 44 1-115 26-35 50-66 99-104 m30E1 45 1-115 26-35 50-6699-104 m10G8 46 1-118 26-35 50-66 99-107 m19E9 47 1-118 26-35 50-6699-107 m10H11 48 1-118 26-35 50-66 99-107

In one embodiment, the antibodies of the present invention or bindingfragments thereof comprise CDRs comprising one of several variable aminoacids at certain positions. In one embodiment antibodies of the presentinvention, or binding fragments thereof, comprise the “CDR Variable”domains listed at SEQ ID NOs: 78-89. These “CDR Variable” sequencesinclude the consensus sequence of each family of related antibodies aswell as variable positions encompassing all observed sequence variantswithin that family. Such sequence variants are displayed in FIGS. 1A-1Cand 2A-2C.

In another embodiment, the variable amino acids in potential CDRs areselected from those amino acids appearing two or more times in thefamilies reported herein. These antibodies are a subset of the “CDRVariable” antibodies described above in which amino acids that appearonly once at a given position in a CDR in a given family of sequencesare not included in the pool of potential CDRs. These “singleoccurrence” amino acid substitutions are readily determined, and thusexcluded from the “CDR Variable” sequences, by simple inspection ofFIGS. 1A-1C and 2A-2C. This narrowed range of potential CDR sequences isreferred to herein as a “multiple occurrence variable CDR.” Thisnomenclature is used herein for convenience in referring to this subsetof the “CDR Variable” sequences.

In yet another embodiment, potential CDRs are not limited to the “CDRVariable” sequences described above, but also include conservativelymodified variants of any observed amino acid, as determined using thedata of Table 1.

In a further embodiment, potential CDRs include variants of any singlesequence CDR disclosed herein, including consensus sequences SEQ ID NOs:66-77, in which the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore conservative amino acid substitutions relative to the disclosedsequence, as determined using the data of Table 1.

Also contemplated are chimeric antibodies. As noted above, typicalchimeric antibodies comprise a portion of the heavy and/or light chainidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

Bispecific antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes, e.g.,IL-23p19 and IL-23p40. In one embodiment, the epitopes are from the sameantigen. In another embodiment, the epitopes are from two differentantigens. Methods for making bispecific antibodies are known in the art.For example, bispecific antibodies can be produced recombinantly usingthe co-expression of two immunoglobulin heavy chain/light chain pairs.See, e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al. (1985) Science 229:81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Hollinger, et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90: 6444-48, Gruber, et al., J. Immunol. 152:5368 (1994).

In yet other embodiments, different constant domains may be appended tothe humanized V_(L) and V_(H) regions provided herein. For example, if aparticular intended use of an antibody (or fragment) of the presentinvention were to call for altered effector functions, a heavy chainconstant domain other than IgG1 may be used. Although IgG1 antibodiesprovide for long half-life and for effector functions, such ascomplement activation and antibody-dependent cellular cytotoxicity, suchactivities may not be desirable for all uses of the antibody. In suchinstances an IgG4 constant domain, for example, may be used.

V. Biological Activity of Humanized Anti-IL-23

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-IL-23 antibody can be screened forinhibitory biologic activity in vitro or suitable binding affinity. Toscreen for antibodies that bind to the epitope on human IL-23 (i.e. thep19 subunit) bound by an antibody of interest (e.g., those which blockbinding of the cytokine to its receptor), a routine cross-blocking assaysuch as that described in ANTIBODIES, A LABORATORY MANUAL, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.Antibodies that bind to the same epitope are likely to cross-block insuch assays, but not all cross-blocking antibodies will necessarily bindat precisely the same epitope since cross-blocking may result fromsteric hindrance of antibody binding by antibodies bound at nearby, oreven overlapping, epitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells (1989) Science 244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human IL-23 may also be used to determine the functionalepitope for an anti-IL-23 antibody of the present invention. Mutagenesisstudies, however, may also reveal amino acid residues that are crucialto the overall three-dimensional structure of IL-23 but that are notdirectly involved in antibody-antigen contacts, and thus other methodsmay be necessary to confirm a functional epitope determined using thismethod.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofhuman IL-23p19 (SEQ ID NO: 39). A series of overlapping peptidesencompassing the sequence of IL-23p19 may be synthesized and screenedfor binding, e.g. in a direct ELISA, a competitive ELISA (where thepeptide is assessed for its ability to prevent binding of an antibody toIL-23p19 bound to a well of a microtiter plate), or on a chip. Suchpeptide screening methods may not be capable of detecting somediscontinuous functional epitopes, i.e. functional epitopes that involveamino acid residues that are not contiguous along the primary sequenceof the IL-23p19 polypeptide chain.

The epitope bound by antibodies of the present invention may also bedetermined by structural methods, such as X-ray crystal structuredetermination (e.g., WO2005/044853), molecular modeling and nuclearmagnetic resonance (NMR) spectroscopy, including NMR determination ofthe H-D exchange rates of labile amide hydrogens in IL-23 when free andwhen bound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993)Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Additional antibodies binding to the same epitope as an antibody of thepresent invention may be obtained, for example, by screening ofantibodies raised against IL-23 for binding to the epitope, or byimmunization of an animal with a peptide comprising a fragment of humanIL-23 comprising the epitope sequence. Antibodies that bind to the samefunctional epitope might be expected to exhibit similar biologicalactivities, such as blocking receptor binding, and such activities canbe confirmed by functional assays of the antibodies.

Antibody affinities (e.g. for human IL-23) may be determined usingstandard analysis. Preferred humanized antibodies are those which bindhuman IL-23p19 with a K_(D) value of no more than about 1×10⁻⁷;preferably no more than about 1×10⁻⁸; more preferably no more than about1×10⁻⁹; and most preferably no more than about 1×10⁻¹⁰ M.

The antibodies and fragments thereof useful in the present compositionsand methods are biologically active antibodies and fragments. As usedherein, the term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired the antigenic epitopeand directly or indirectly exerting a biologic effect. Typically, theseeffects result from the failure of IL-23 to bind its receptor. As usedherein, the term “specific” refers to the selective binding of theantibody to the target antigen epitope. Antibodies can be tested forspecificity of binding by comparing binding to IL-23 to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to IL-23 at least 10, and preferably 50 times morethan to irrelevant antigen or antigen mixture then it is considered tobe specific. An antibody that “specifically binds” to IL-23 does notbind to proteins that do not comprise the IL-23-derived sequences, i.e.“specificity” as used herein relates to IL-23 specificity, and not anyother sequences that may be present in the protein in question. Forexample, as used herein, an antibody that “specifically binds” to IL-23will typically bind to FLAG-hIL-23, which is a fusion protein comprisingIL-23 and a FLAG® peptide tag, but it does not bind to the FLAG® peptidetag alone or when it is fused to a protein other than IL-23.

IL-23-specific binding compounds of the present invention, such asinhibitory IL-23p19 specific antibodies, can inhibit its biologicalactivity in any manner, including but not limited to production of IL-1βand TNF by peritoneal macrophages and IL-17 by T_(H)17 T cells (seeLangrish et al. (2004) Immunol. Rev. 202:96-105). Anti-IL-23p19antibodies will also be able to inhibit the gene expression of IL-17A,IL-17F, CCL7, CCL17, CCL20, CCL22, CCR1, and GM-CSF (see Langrish et al.(2005) J. Exp. Med. 201:233-240). IL-23-specific binding compounds ofthe present invention, such as anti IL-23p19 antibodies, will also blockthe ability of IL-23 to enhance proliferation or survival of T_(H)17cells. Cua and Kastelein (2006) Nat. Immunol. 7:557-559. The inhibitoryactivity of engineered anti-IL-23p19 will be useful in the treatment ofinflammatory, autoimmune, and proliferative disorders. Such disordersare described in PCT patent application publications WO 04/081190; WO04/071517; WO 00/53631; and WO 01/18051, the disclosures of which arehereby incorporated by reference in their entireties.

VI. Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions including an agonistor antagonist of IL-23, the cytokine analogue or mutein, antibodythereto, or nucleic acid thereof, is admixed with a pharmaceuticallyacceptable carrier or excipient, see, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical DosageForms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio between LD₅₀ and ED₅₀. Antibodies exhibiting high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein human. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

The mode of administration is not particularly important. Suitableroutes of administration may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof antibody used in the pharmaceutical composition or to practice themethod of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral,intraarterial or intravenous injection. Intravenous administration tothe patient is preferred.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003)New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, etal. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl.J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing an inflammatory, autoimmune, orproliferative response to the reagent.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A preferred doseprotocol is one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects. A total weekly dose is generallyat least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, mostgenerally at least 0.5 μg/kg, typically at least 1 μg/kg, more typicallyat least 10 μg/kg, most typically at least 100 μg/kg, preferably atleast 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably atleast 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at least 25mg/kg, and most optimally at least 50 mg/kg (see, e.g., Yang, et al.(2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J.Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother.52:133-144). The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with autoimmunedisease or pathogen-induced immunopathology and/or a reduction in theseverity of such symptoms that will or are expected to develop. Theterms further include ameliorating existing uncontrolled or unwantedautoimmune-related or pathogen-induced immunopathology symptoms,preventing additional symptoms, and ameliorating or preventing theunderlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with anautoimmune or pathogen-induced immunopathology disease or symptom, orwith the potential to develop such a disease or symptom.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an IL-23p19 specific bindingcompound, e.g. and antibody, that when administered alone or incombination with an additional therapeutic agent to a cell, tissue, orsubject is effective to prevent or ameliorate the autoimmune disease orpathogen-induced immunopathology associated disease or condition or theprogression of the disease. A therapeutically effective dose furtherrefers to that amount of the compound sufficient to result inamelioration of symptoms, e.g., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient administered alone, atherapeutically effective dose refers to that ingredient alone. Whenapplied to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of therapeutic will decrease thesymptoms typically by at least 10%; usually by at least 20%; preferablyat least about 30%; more preferably at least 40%, and most preferably byat least 50%.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art, see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa. The pharmaceutical composition of the invention mayalso contain other immunosuppressive or immunomodulating agents. Anysuitable immunosuppressive agent can be employed, including but notlimited to anti-inflammatory agents, corticosteroids, cyclosporine,tacrolimus (i.e., FK-506), sirolimus, interferons, soluble cytokinereceptors (e.g., sTNRF and sIL-1R), agents that neutralize cytokineactivity (e.g., inflixmab, etanercept), mycophenolate mofetil,15-deoxyspergualin, thalidomide, glatiramer, azathioprine, leflunomide,cyclophosphamide, methotrexate, and the like. The pharmaceuticalcomposition can also be employed with other therapeutic modalities suchas phototherapy and radiation.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

VII. Antibody Production

For recombinant production of the antibodies of the present invention,the nucleic acids encoding the two chains are isolated and inserted intoone or more replicable vectors for further cloning (amplification of theDNA) or for expression. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. In one embodiment, both thelight and heavy chains of the humanized anti-IL-23p19 antibody of thepresent invention are expressed from the same vector, e.g. a plasmid oran adenoviral vector.

Antibodies of the present invention may be produced by any method knownin the art. In one embodiment, antibodies are expressed in mammalian orinsect cells in culture, such as chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, babyhamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9) cells.In one embodiment, antibodies secreted from CHO cells are recovered andpurified by standard chromatographic methods, such as protein A, cationexchange, anion exchange, hydrophobic interaction, and hydroxyapatitechromatography. Resulting antibodies are concentrated and stored in 20mM sodium acetate, pH 5.5.

In another embodiment, the antibodies of the present invention areproduced in yeast according to the methods described in WO2005/040395.Briefly, vectors encoding the individual light or heavy chains of anantibody of interest are introduced into different yeast haploid cells,e.g. different mating types of the yeast Pichia pastoris, which yeasthaploid cells are optionally complementary auxotrophs. The transformedhaploid yeast cells can then be mated or fused to give a diploid yeastcell capable of producing both the heavy and the light chains. Thediploid strain is then able to secret the fully assembled andbiologically active antibody. The relative expression levels of the twochains can be optimized, for example, by using vectors with differentcopy number, using transcriptional promoters of different strengths, orinducing expression from inducible promoters driving transcription ofthe genes encoding one or both chains.

In one embodiment, the respective heavy and light chains of a pluralityof different anti-IL-23p19 antibodies (the “original” antibodies) areintroduced into yeast haploid cells to create a library of haploid yeaststrains of one mating type expressing a plurality of light chains, and alibrary of haploid yeast strains of a different mating type expressing aplurality of heavy chains. These libraries of haploid strains can bemated (or fused as spheroplasts) to produce a series of diploid yeastcells expressing a combinatorial library of antibodies comprised of thevarious possible permutations of light and heavy chains. Thecombinatorial library of antibodies can then be screened to determinewhether any of the antibodies has properties that are superior (e.g.higher affinity for IL-23) to those of the original antibodies.See.e.g., WO2005/040395.

In another embodiment, antibodies of the present invention are humandomain antibodies in which portions of an antibody variable domain arelinked in a polypeptide of molecular weight approximately 13 kDa. See,e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, lowmolecular weight agents provide numerous advantages in terms of ease ofsynthesis, stability, and route of administration.

VIII. Uses

The present invention provides methods for using engineered anti-IL-23for the treatment and diagnosis of inflammatory disorders andconditions, e.g., of the central nervous system, peripheral nervoussystem, and gastrointestinal tract, as well as autoimmune andproliferative disorders.

Methods are provided for the treatment of, e.g., multiple sclerosis(MS), including relapsing-remitting MS and primary progressive MS,Alzheimer's disease, amyotrophic lateral sclerosis (a.k.a. ALS; LouGehrig's disease), ischemic brain injury, prion diseases, andHIV-associated dementia. Also provided are methods for treatingneuropathic pain, posttraumatic neuropathies, Guillain-Barre syndrome(GBS), peripheral polyneuropathy, and nerve regeneration.

Provided are methods for treating or ameliorating one or more of thefollowing features, symptoms, aspects, manifestations, or signs ofmultiple sclerosis, or other inflammatory disorder or condition of thenervous system: brain lesions, myelin lesions, demyelination,demyelinated plaques, visual disturbance, loss of balance orcoordination, spasticity, sensory disturbances, incontinence, pain,weakness, fatigue, paralysis, cognitive impairment, bradyphrenia,diplopia, optic neuritis, paresthesia, gait ataxia, fatigue, Uhtoff'ssymptom, neuralgia, aphasia, apraxia, seizures, visual-field loss,dementia, extrapyramidal phenomena, depression, sense of well-being, orother emotional symptoms, chronic progressive myelopathy, and a symptomdetected by magnetic resonance imaging (MRI), includinggadolinium-enhancing lesions, evoked potential recordings, orexamination of cerebrospinal fluid (see, e.g., Kenealy et al. (2003) J.Neuroimmunol. 143:7-12; Noseworthy et al. (2000) New Engl. J. Med.343:938-952; Miller et al. (2003) New Engl. J. Med. 348:15-23; Chang etal. (2002) New Engl. J. Med. 346:165-173; Bruck and Stadelmann (2003)Neurol. Sci. 24 Suppl. 5:S265-S267).

Moreover, the present invention provides methods for treating anddiagnosing inflammatory bowel disorders, e.g., Crohn's disease,ulcerative colitis, celiac disease, and irritable bowel syndrome.Provides are methods for treating or ameliorating one or more of thefollowing symptoms, aspects, manifestations, or signs of an inflammatorybowel disorder: malabsorption of food, altered bowel motility,infection, fever, abdominal pain, diarrhea, rectal bleeding, weightloss, signs of malnutrition, perianal disease, abdominal mass, andgrowth failure, as well as intestinal complications such as stricture,fistulas, toxic megacolon, perforation, and cancer, and includingendoscopic findings, such as, friability, aphthous and linear ulcers,cobblestone appearance, pseudopolyps, and rectal involvement and, inaddition, anti-yeast antibodies (see, e.g., Podolsky, supra; Hanauer,supra; Horwitz and Fisher, supra).

Also contemplated is treatment of inflammatory disorders such aspsoriasis, atopic dermatitis, arthritis, including rheumatoid arthritis,osteoarthritis, and psoriatic arthritis, autoimmune disorders, such assystemic lupus erythematosus and type I diabetes, and proliferativedisorders such as cancer (see, e.g., PCT patent applications WO04/081190; WO04/071517; WO00/53631; and WO01/18051).

The IL-23p19 binding compounds of the present invention can also be usedin combination with one or more antagonists of other cytokines (e.g.antibodies), including but not limited to, IL-17A, IL-1β, IL-6 andTGF-β. See, e.g., Veldhoen (2006) Immunity 24:179-189; Dong (2006) Nat.Rev. Immunol. 6(4):329-333. In various embodiments, an IL-23p19 bindingcompound of the invention is administered before, concurrently with, orafter administration of the another antagonist or antagonists, such asan anti-IL-17A antibody. In one embodiment, an IL-17A binding compoundis used in treatment of the acute early phase of an adverse immuneresponse (e.g. MS, Crohn's Disease) alone or in combination with anIL-23 antagonist antibody of the present invention. In the latter case,the IL-17A binding compound may be gradually decreased and treatmentwith the antagonist of IL-23 alone is continued to maintain suppressionof the adverse response. Alternatively, antagonists to IL-1β, IL-6and/or TGF-β may be administered concurrently, before or after anIL-23p19 binding compound of the present invention. See Cua andKastelein (2006) Nat. Immunol. 7:557-559; Tato and O'Shea (2006) Nature441:166-168; Iwakura and Ishigame (2006) J. Clin. Invest. 116:1218-1222.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

EXAMPLES Example 1 General Methods

Standard methods in molecular biology are described (Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif.). Standard methods also appear in Ausbel et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, JohnWiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocolsin Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described(Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra). Standard techniques for characterizing ligand/receptorinteractions are available (see, e.g., Coligan et al. (2001) CurrentProtocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742;Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren etal. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

Example 2 Humanization of Anti-Human IL-23p19 Antibodies

The humanization of mouse anti-human IL-23p19 antibodies 6H12 and 7G10,was performed as essentially as described in PCT patent applicationpublications WO 2005/047324 and WO 2005/047326, which are incorporatedby reference.

Variable light and heavy domains of selected anti-IL-23 monoclonalantibodies (6H12 and 7G10) were cloned and fused to a human kappa lightchain (CL domain) and human IgG1 heavy chain (CH1-hinge-CH2-CH3),respectively.

The amino acid sequence of the non-human VH domain was compared to agroup of five human VH germline amino acid sequences; one representativefrom subgroups IGHV1 and IGHV4 and three representatives from subgroupIGHV3. The VH subgroups are listed in M.-P. Lefranc, “Nomenclature ofthe Human Immunoglobulin Heavy (IGH) Genes”, Experimental and ClinicalImmunogenetics, 18:100-116, 2001. 6H12 and 7G10 antibodies scoredhighest against human heavy chain germline DP-14 in subgroup VH1.

For all non-human antibodies, the VL sequences were of the kappasubclass of VL. The amino acid sequence of the non-human VL domain wascompared to a group of four human VL kappa germline amino acidsequences. The group of four is comprised of one representative fromeach of four established human VL subgroups listed in V. Barbie & M.-P.Lefranc, “The Human Immunoglobulin Kappa Variable (IGKV) Genes andJoining (IGKJ) Segments”, Experimental and Clinical Immunogenetics,15:171-183, 1998 and M.-P. Lefranc, “Nomenclature of the HumanImmunoglobulin Kappa (IGK) Genes”, Experimental and ClinicalImmunogenetics, 18:161-174, 2001. The four subgroups also correspond tothe four subgroups listed in Kabat et al. “Sequences of Proteins ofImmunological Interest”, U.S. Department of Health and Human Services,NIH Pub. 91-3242, 5th Ed., 1991, pp. 103-130. 6H12 and 7G10 antibodiesscored highest against human light chain germline Z-012 in subgroupVLkI.

Once the target amino acid sequences of the variable heavy and lightchains were determined, plasmids encoding the full-length humanizedantibody were generated. Starting with a plasmid encoding a humanizedanti-IL-10 antibody having VH3 DP-46 and VLkI Z-012 germline frameworks,the plasmids were altered using Kunkel mutagenesis (see, e.g., Kunkel TA. (1985) Proc. Natl. Acad. Sci. U.S.A 82:488-492) to change the DNAsequence to the target humanized 6H12 or 7G10 sequences. Simultaneously,codon optimization was incorporated into the changes to provide forpotentially optimal expression. The resulting humanized heavy and lightchain sequences, including signal sequences, are provided at SEQ ID NOs:1 and 2 (antibody 6H12) and at SEQ ID NOs: 3 and 4 (for antibody 7G10),respectively.

An analogous procedure was performed to determine the proper humanframeworks for humanization of antibodies 10H11 and 22E9. Antibody 10H11scored highest against human antibody heavy chain germline DP-46 insubgroup VH3 and light chain germline Z-A27 in subgroup VLkIII. Antibody22E9 scored highest against human antibody heavy chain germline DP-14 insubgroup VH1 and light chain germline Z-B3 in subgroup VLkIV. Theresulting humanized heavy and light chain variable domain sequences areprovided at SEQ ID NOs: 90 and 91 (antibody 10H11) and at SEQ ID NOs: 92and 93 (for antibody 22E9), respectively.

Example 3 Determining the Equilibrium Dissociation Constant (K_(D)) forHumanized Anti-Human IL-23 Using KinExA Technology

The equilibrium dissociation constant (K_(D)) for anti human IL-23antibodies were determined using the KinExA 3000 instrument (SapidyneInstruments Inc., www.sapidyne.com). KinExA uses the principle of theKinetic Exclusion Assay method based on measuring the concentration ofuncomplexed antibody in a mixture of antibody, antigen andantibody-antigen complex. The concentration of free antibody is measuredby exposing the mixture to a solid-phase immobilized antigen for a verybrief period of time. In practice, this is accomplished by flowing thesolution phase antigen-antibody mixture past antigen-coated particlestrapped in a flow cell. Data generated by the instrument are analyzedusing custom software. Equilibrium constants are calculated using amathematical theory based on the following assumptions:

1. The binding follows the reversible binding equation for equilibrium:k _(on) [Ab][Ag]=k _(off) [AbAg]

2. Antibody and antigen bind 1:1 and total antibody equalsantigen-antibody complex plus free antibody.

3. Instrument signal is linearly related to free antibody concentration.

98 micron PMMA particles (Sapidyne, Cat No. 440198) were coated withbiotinylated rhIL-23 according to Sapidyne “Protocol for coating PMMAparticles with biotinylated ligands having short or nonexistent linkerarms”. For biotinylation of rhIL-23, EZ-link TFP PEO-biotin (Pierce,Cat. No. 21219) was used according to manufacturer's recommendations(Pierce bulletin 0874). All experimental procedures were done accordingto the KinExA 3000 manual.

Three forms of the heterodimeric IL-23 protein were used. Native ornon-linked human IL-23 comprised of two chains, p19 and p40, that arecovalently linked by a disulfide-bond. “Non-linked” IL-23 is comprisedof human p40 coexpressed in 293T cells with human p19:FLAG-tag peptideand purified over an anti-FLAG peptide affinity column. By non-reducingSDS-PAGE the purified non-linked IL-23 showed presence of multimericIL-23 forms at molecular weights corresponding to dimers, trimers, etc.

“Elastikine” IL-23 is a single-chain peptide comprised of FLAG-tagpeptide:GLU-tag peptide:human p40:elasti-linker:human p19. Theelasti-linker peptide sequence was derived from R&D Systems form ofcommercial IL-23. Elastikine was expressed in 293T cells and purifiedover an anti-FLAG peptide affinity column. By non-reducing SDS-PAGE thepurified elastikine IL-23 showed presence of multimeric IL-23 forms atmolecular weights corresponding to dimers, trimers, etc.

A non-tagged, non-linked form of native human IL-23p19/p40 coexpressedin SF9 cells was purchased from eBioscience (CAT No. 34-8239). Bynon-reducing SDS-PAGE the purified eBioscience human IL-23 did not showpresence of multimeric IL-23 forms.

All runs were done in duplicate under the following conditions: Samplevolume: 1.5 mL; Sample flow rate: 0.25 mL/min; Label volume: 0.5 mL;Label flow rate: 0.25 ml/min; mAb conc.: 0.1 nM; Highest Ag (hIL-23)conc.: 4.0 nM; Lowest Ag (hIL-23) conc.: 3.91 pM. Two-fold serialdilutions of the antigen were prepared and mixed with the antibody atconstant concentration. The mixture was incubated for 2 hr at roomtemperature to equilibrate.

Table 4 shows the results of the KinExA analysis.

TABLE 4 K_(D) Values Determined by KinExa Human IL-23 Antibody K_(D)(pM)elastikine 6H12 54, 48 non-linked 6H12 >1200 eBioscience6H12 >1000, >920  elastikine hu6H12 28, 36 elastikine 7G10  41, 9.2elastikine hu7G10 49, 16 elastikine 39G2 19 non-linked 39G2 34eBioscience 39G2 620 elastikine 35F12 53 eBioscience 35F12 >700elastikine 13B5 22 eBioscience 13B5 55 elastikine 7D7 2.7 elastikine 3D70.84 elastikine 49A10 7.4 elastikine 13F11 11 elastikine 33B12 6.8

Example 4 Determining the Equilibrium Dissociation Constant (K_(D)) forHumanized Anti-Human IL-23p19 Antibodies Using BIAcore Technology

All ligands (anti-IL-23 mAbs) were immobilized on a BIAcore CM5 sensorchip using standard amine-coupling procedure. All experiments werecarried out at 25° C. and at a flow-rate of 10 μL/min in PBS. All IL-23forms were diluted in PBS to produce various concentrations. Kineticconstants for the various interactions were determined usingBIAevaluation software 3.1. The K_(D) was determined using thecalculated dissociation and association rate constants. Proteins wereused at the following concentrations: anti-IL-23 mAb hu7G10 in PBS at0.33 mg/mL; anti-IL-23 mAb hu6H12 in PBS at 0.2 mg/mL; bac-wt humanIL-23 in PBS at 0.30 mg/mL; eBioscience human IL-23 in PBS at 0.10mg/mL; N222Q human IL-23 in PBS at 0.33 mg/mL.

In addition to the proteins noted above other forms were also used.“Bac-wt” human IL-23 is identical to “elastikine” human IL-23 insequence. This IL-23 was expressed in SF9 cells and purified over ananti-FLAG peptide affinity column. By non-reducing SDS-PAGE the purifiedIL-23 did not show presence of multimeric IL-23 forms. “N222Q” humanIL-23 is identical to “elastikine” human IL-23 in sequence except foralteration of Asn222 to Gln in the p40 subunit (GenBank Accession No.P29460). This IL-23 was expressed in SF9 cells and purified over ananti-FLAG peptide affinity column. By non-reducing SDS-PAGE the purifiedN222Q I-23 did not show presence of multimeric IL-23 forms.

The following immobilization and regeneration conditions were used forall experiments: Flow-rate: 5 μL/min; NHS/EDC: 10 μL; Protein: 5 μg/mLin 10 mM NaAcetate, pH 5.0:10 μL; ethanolamine: 40 μL; regeneration: 5μL of 50 mM NaOH.

Table 5 provides the K_(D) values as determined by BIAcore.

TABLE 5 K_(D) Determination by BIAcore Human IL-23 Antibody K_(D)(nM)bac-wt hu7G10 10   N222Q hu7G10 0.3, 1.0 eBioscience hu7G10 3.2, 9.0bac-wt hu6H12 5.1 N222Q hu6H12 0.5 eBioscience hu6H12 4.1

Example 5 Proliferation Bioassays for the Assessment of NeutralizingAnti-IL-23 Antibodies

The ability of a monoclonal antibody to biologically neutralize IL-23was assessed by the application of short-term proliferation bioassaysthat employ cells that express recombinant IL-23 receptors. Thetransfectant Ba/F3-2.2lo cells proliferate in response to human IL-23and the response can be inhibited by a neutralizing anti-IL-23 antibody.An antibody is titrated against a concentration of IL-23 chosen withinthe linear region of the dose-response curve, near plateau and aboveEC50. Proliferation, or lack thereof, is measured by colorimetric meansusing Alamar Blue, a growth indicator dye based on detection ofmetabolic activity. The ability of an antibody to neutralize IL-23 isassessed by its IC50 value, or concentration of antibody that induceshalf-maximal inhibition of IL-23 proliferation.

IL-23R Transfectant Cell Line and Cell Culture Cell line IL-23Rexpression IL-23 response Ba/F3-2.2lo-hIL-23R hIL-23R, hIL-12RB1 Human,Cyno

Ba/F3 transfectants are maintained in RPMI-1640 medium, 10% fetal calfserum, 50 μM 2-mercaptoethanol, 2 mM L-Glutamine, 50 μg/mLpenicillin-streptomycin, and 10 ng/mL mouse IL-3. “Cyno” refers tocynomolgus monkey IL-23.

Proliferation Bioassay Medium

Ba/F3 proliferation bioassays were performed in RPMI-1640 medium, 10%fetal calf serum, 50 uM 2-mercaptoethanol, 2 mM L-Glutamine, and 50ug/mL penicillin-streptomycin.

Procedure

Assays were performed in 96-well flat bottom plates (Falcon 3072 orsimilar). All preparations of reagents and cell suspensions utilized theappropriate bioassay medium. The assay volume was 150 μL per well.Titrations of an anti-IL-23 antibody were pre-incubated with IL-23 for30-60 min at room temperature, during which time cells were prepared.Cells were added to plates following the antibody-cytokinepre-incubation. Bioassay plates were incubated in a humidified tissueculture chamber (37C, 5% CO₂) for 40-48 hr. At the end of the culturetime, Alamar Blue (Biosource Cat #DAL1100) was added at 16.5 μL/well andallowed to develop for 5-12 hours. Absorbance was then read at 570 nmand 600 nm (VERSAmax Microplate Reader, Molecular Probes), and anOD₅₇₀₋₆₀₀ was obtained. Duplicates were run for each sample.

Cell Preparation

Cells were used in a healthy growth state, generally at densities of3-8×10⁵/mL. Cells were counted, pelleted, washed twice in bioassaymedium, and suspended to the appropriate density for plating.

IL-23 Dose-Response

IL-23 was prepared to working concentration (3 ng/mL) and added to firstwell at 75 μL. Serial dilutions of 1:3 were made by titrating 25:50 μLin bioassay medium across wells, leaving 50 μL/well. Cells weresuspended to the appropriate density for plating at 100 μL per well.

Neutralizing Antibody Dose-Response

The antibody was prepared to working concentration (30 μg/mL) and addedto first well at 75 μL. Serial dilutions of 1:3 were made by titrating25:50 μL in bioassay medium across wells, leaving 50 μL per well. IL-23at the appropriate concentration was added at 50 μL per well to thewells containing the titrated antibody. Cells were suspended to theappropriate density for plating at 50 μL per well, and added followingthe antibody-cytokine pre-incubation.

IC50 Determination

Using GraphPad Prism 3.0 software, absorbance is plotted againstcytokine or antibody concentration and IC50 values are determined usingnon-linear regression (curve fit) of sigmoidal dose-response.

Table 6 shows the IC50 values for blocking of Ba/F3 cell proliferationby anti-IL-23p19 antibodies

TABLE 6 IC50 Values for Blocking of Ba/F3 Cell Proliferation byAnti-IL-23 Antibodies Human IL-23 Antibody IC50(nM) elastikine 7G10 22,18 non-linked 7G10 3000 eBioscience 7G10 3100, 510  elastikine hu7G10 29 non-linked hu7G10 10000  eBioscience hu7G10 7800 elastikine 6H12  9,11 non-linked 6H12 1500 eBioscience 6H12 1300, 500  elastikine hu6H12 27 non-linked hu6H12 4000 eBioscience hu6H12 3200 elastikine 13B5 7, 5non-linked 13B5  113 eBioscience 13B5  31 elastikine 33B12 4, 3non-linked 33B12  193 eBioscience 33B12  57 elastikine 39G2 9, 5non-linked 39G2  67 eBioscience 39G2  11 elastikine 35F12 15, 5 non-linked 35F12  73 eBioscience 35F12  12 elastikine 3D7 3, 3non-linked 3D7  37 eBioscience 3D7   2

Example 6 Epitope for Anti-IL-23p19 Antibody 7G10

The epitope for the binding of antibody 7G10 to human IL-23p19 (SEQ IDNO: 29) was determined by X-ray crystallography. Coordinates weredetermined for a complex of an Fab fragment of the chimeric form ofantibody 7G10 and non-linked human IL-23, which comprises p19 and p40subunits. The sequence of human IL-23p19 is found at SEQ ID NO: 29 andthe sequence of the mature form of human IL-12/IL-23 p40 is found atresidues 23-328 of GenBank Accession No. P29460. The chimeric form ofantibody 7G10 comprises i) a heavy chain comprising the mouse 7G10 V_(H)domain (SEQ ID NO: 6) fused to a human heavy chain constant region(residues 135-464 of SEQ ID NO: 3), and ii) a light chain comprising themouse 7G10 V_(L) domain (SEQ ID NO: 18) fused to a human light chainconstant region (residues 130-233 of SEQ ID NO: 4).

IL-23 amino acid residues within 4.0 Å of residues on antibody 7G10include E82, G86, S87, D88, T91, G92, E93, P94, S95, H106, P133, S134,Q135, P136, W137, R139, L140. Additional residues K83, F90 and L110 werewithin 5.0 Å. An amino acid residue on IL-23p19 is considered to bewithin a given distance of the antibody (e.g. 4.0 Å or 5.0 Å) if thecoordinates of any atom of the residue are within the given distance ofthe coordinates of any atom of the antibody.

Most of these contacted residues fall into two main clusters along theprimary structure of IL-23p19, with the first cluster comprisingresidues 82-95 (in which 11 of 14 residues are within 5.0 Å of theantibody and 9 of 14 are within 4.0 Å) and the second cluster comprisingresidues 133-140 (in which 7 of 8 residues are within 4.0 Å of theantibody). These clusters define epitopes comprising stretches of 8 ormore contiguous amino acid residues of IL-23p19 in which 50%, 70% and85% or more of the residues are within 5.0 Å of the antibody.

Antibodies binding to either or both of these clusters would be expectedto block binding of antibody 7G10. Given the strong sequence homologybetween all six CDR sequences (see FIGS. 1A-2C), it is likely that theother antibodies comprising the “(a) light chain subfamily” (6H12,33B12, 13F11, 13B5, 13G1, 11C10, 7E2, 30F11, 34E4, 6H4, 33D2) will alsobind to substantially the same epitope in IL-23p19 as antibody 7G10. Theconsensus CDR sequences for the antibodies of the “(a) light chainsubfamily” variable domain sequence are provided at SEQ ID NOs: 69, 72and 75. Corresponding heavy chain variable domain consensus sequencesare provided at SEQ ID NOs: 66-68. Antibodies binding to the sameepitope as antibody 7G10 would be expected to exhibit similar biologicalactivities, such as blocking Ba/F3 cell proliferation in the assaydescribed at Example 5 and Table 6, albeit with perhaps somewhatvariable affinities and IC50s.

Table 7 provides a brief description of the sequences in the sequencelisting.

TABLE 7 Sequence Identifiers SEQ ID NO: Description 1 hum6H12 HC 2hum6H12 LC 3 hum7G10 HC 4 hum7G10 LC 5 m6H12 V_(H) 6 m7G10 V_(H) 7m13F11 V_(H) 8 m13B5 V_(H) 9 m21A10 V_(H) 10 m33B12 V_(H) 11 m39G2 V_(H)12 m35F12 V_(H) 13 m49A10 V_(H) 14 m3D7 V_(H) 15 m34F9 V_(H) 16 m7D7V_(H) 17 m6H12 V_(L) 18 m7G10 V_(L) 19 m13F11 V_(L) 20 m13B5 V_(L) 21m21A10 V_(L) 22 m33B12 V_(L) 23 m39G2 V_(L) 24 m35F12 V_(L) 25 m49A10V_(L) 26 m34F9 V_(L) 27 m7D7 V_(L) 28 m3D7 V_(L) 29 Human IL23p19 30Murine IL-23p19 31 m13G1 V_(H) 32 m11C10 V_(H) 33 m7E2 V_(H) 34 m30F11V_(H) 35 m34E4 V_(H) 36 m6H4 V_(H) 37 m33D2 V_(H) 38 m1E10 V_(H) 39m20A9 V_(H) 40 m22E9 V_(H) 41 m29D5 V_(H) 42 m5B12 V_(H) 43 m9C9 V_(H)44 m11B10 V_(H) 45 m30E1 V_(H) 46 m10G8 V_(H) 47 m19E9 V_(H) 48 m10H11V_(H) 49 m13G1 V_(L) 50 m11C10 V_(L) 51 m7E2 V_(L) 52 m30F11 V_(L) 53m34E4 V_(L) 54 m6H4 V_(L) 55 m33D2 V_(L) 56 m1E10 V_(L) 57 m20A9 V_(L)58 m22E9 V_(L) 59 m29D5 V_(L) 60 m5B12 V_(L) 61 m9C9 V_(L) 62 m11B10V_(L) 63 m10G8 V_(L) 64 m19E9 V_(L) 65 m10H11 V_(L) 66 CDR-H1 Consensus67 CDR-H2 Consensus 68 CDR-H3 Consensus 69 CDR-L1(a) Consensus 70CDR-L1(b) Consensus 71 CDR-L1(c) Consensus 72 CDR-L2(a) Consensus 73CDR-L2(b) Consensus 74 CDR-L2(c) Consensus 75 CDR-L3(a) Consensus 76CDR-L3(b) Consensus 77 CDR-L3(c) Consensus 78 CDR-H1 Variable 79 CDR-H2Variable 80 CDR-H3 Variable 81 CDR-L1(a) Variable 82 CDR-L1(b) Variable83 CDR-L1(c) Variable 84 CDR-L2(a) Variable 85 CDR-L2(b) Variable 86CDR-L2(c) Variable 87 CDR-L3(a) Variable 88 CDR-L3(b) Variable 89CDR-L3(c) Variable 90 hum10H11 V_(H) 91 hum10H11 V_(L) 92 hum22E9 V_(H)93 hum22E9 V_(L)

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

Citations of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. U.S. patents and other publicationsreferenced herein are hereby incorporated by reference.

1. A purified monoclonal antibody, or antigen binding fragment thereof,that binds to human IL-23p19 at an epitope comprising residues 133-140of SEQ ID NO:
 29. 2. A purified monoclonal antibody, or antigen bindingfragment thereof, that binds to human IL-23p19 at an epitope comprisingresidues 82-95 and residues 133-140 of SEQ ID NO:
 29. 3. A purifiedmonoclonal antibody, or antigen binding fragment thereof, that is ableto block the binding of a binding compound to human IL-23p19 in across-blocking assay, wherein the binding compound consists of: amonoclonal antibody, or antigen binding fragment thereof, that binds tohuman IL-23p19 at an epitope comprising residues 133-140 of SEQ ID NO:29.
 4. A purified monoclonal antibody, or antigen binding fragmentthereof, that is able to block the binding of a binding compound tohuman IL-23p19 in a cross-blocking assay, wherein the binding compoundconsists of: a monoclonal antibody, or antigen binding fragment thereof,that binds to human IL-23p19 at an epitope comprising residues 82-95 andresidues 133-140 of SEQ ID NO: 29.